Antimicrobial PeptideâPolymer Conjugates with High Activity

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Antimicrobial peptide-polymer conjugates with high activity: Influence of polymer molecular weight and peptide sequence on antimicrobial activity, proteolysis and biocompatibility Prashant Kumar, Allen Takayesu, Usama Abbasi, Manu Thomas Kalathottukaren, Srinivas Abbina, Jayachandran N Kizhakkedathu, and Suzana K. Straus ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09471 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 12, 2017

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ACS Applied Materials & Interfaces

Revision of Manuscript ID: am-2017-09471v.R1

9-Oct-17

Antimicrobial peptide-polymer conjugates with high activity: Influence of polymer molecular weight and peptide sequence on antimicrobial activity, proteolysis and biocompatibility

Prashant Kumar1,2, Allen Takayesu1, Usama Abbasi2, Manu Thomas Kalathottukaren2, Srinivas Abbina1,2, Jayachandran N. Kizhakkedathu1,2*, Suzana K. Straus1*

1

Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC,

CANADA V6T 1Z1 2

Centre for Blood Research, Department of Pathology and Laboratory Medicine, University of British

Columbia, 2350 Health Sciences Mall, Life Sciences Centre, Vancouver, BC, CANADA V6T 1Z3

* To whom correspondence should be addressed: J.N. Kizhakkedathu, Phone: 604-822-7085, Fax: 604822-7742, Email:[email protected]; S.K. Straus, Phone: 604-822-2537, Fax: 604-822-2847, Email: [email protected]

Keywords: Hyperbranched polyglycerol, biocompatibility, antimicrobial peptide-polymer conjugates, aurein peptides, bioconjugation, antimicrobial peptides, proteolysis.

1

ACS Paragon Plus Environment


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ABSTRACT We report the synthesis, characterization, activity and biocompatibility of a novel series of antimicrobial peptide-polymer conjugates. Using the parent peptide aurein 2.2, we designed a peptide array (~100 peptides) with single and multiple W and R mutations and identified antimicrobial peptides (AMPs) with potent activity against S. aureus. These novel AMPs were conjugated to hyperbranched polyglycerol (HPG) of different molecular weights and number of peptides to improve their antimicrobial activity and toxicity. The cell and blood compatibility studies of these conjugates demonstrated better properties than the AMP alone. However, conjugates showed lower antimicrobial activity in comparison to peptides, as determined from minimal inhibition concentrations (MICs) against Staphylococcus aureus but considerably better than the available polymer-AMP conjugates in the literature. In addition to measuring MICs and characterizing the biocompatibility, CD spectroscopy was used to investigate the interaction of the novel conjugates with model bacterial biomembranes. Moreover, the novel conjugates were exposed to trypsin to evaluate their stability. It was found that the conjugates resist proteolysis in comparison to unprotected peptides. The peptide-conjugates were active in serum and whole blood. Overall, the results show that combining a highly active AMP and low molecular weight HPG yields bioconjugates with excellent biocompatibility, MICs below 100 µg/ml, and proteolytic stability which could potentially improve its utility for in vivo applications.

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INTRODUCTION Bacterial resistance has forced scientists all over the world to look for alternatives to currently used antibiotics1. Many strategies are being explored2–4, including using antimicrobial peptides (AMPs). Since AMPs have a unique mechanism of action against bacteria, there is little chance of developing resistance towards them5,6. In addition, they have a broad antimicrobial spectrum and some of them are highly potent5,6.

Due to these unique properties, they are hence touted as viable alternatives to

commonly-used antibiotics. Moreover, AMPs are found in secretions of plants and animals and are involved in the first line of defense.

5,7,8

. In spite of their unique features, only a few AMPs are

evaluated for systemic therapy possibly due to poor drug properties, including rapid blood clearance, protease cleavage susceptibility and host cell toxicity9–11. Arenicin and the lantibiotics MU1140 and NVB302 are some of the examples of AMPs used in clinical trials. Significant research is currently being undertaken to address these adverse effects, including the conjugation of AMPs on to polymers and developing controlled release formulations12–15. One approach used for improving the properties of AMPs was the conjugation of polyethylene glycol (PEG) to improve the tissue distribution and efficacy in vivo. Examples of AMPs that have been conjugated to PEG include magainin 210, tachyplesin 19 and nisin16. But in all these examples, cytotoxicity was reduced and the activity of the peptides decreased considerably ranging from the generation of inactive peptides to peptides with low activity (5-64-fold decrease).9,10,16 In addition, the use of linear PEG has important drawbacks such as its high intrinsic viscosity in aqueous conditions that increases with increase in molecular weight, and the lack of multiple functionality. Finally, in some examples, PEGylation did not circumvent the problem of rapid kidney clearance17. Hence, a need to develop alternative approaches arose. To circumvent the limitations associated with PEG conjugation, researchers have used other polymeric systems to develop AMP conjugates with improved efficacy and biocompatibility18. We have 3



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recently demonstrated that hyperbranched polyglycerol (HPG), a highly biocompatible, branched multifunctional polymer20-22, is an ideal scaffold for improving both activity and biocompatibility of AMPs19. HPG has been used recently for the development of drug delivery systems9,12, long circulating drug conjugates20,21, anticoagulant neutralizing agents22,23, for cell surface modification24, cell preservation25 and as an osmotic agent in peritoneal dialysis26. In our published conjugation study, antimicrobial peptide aurein 2.2 was covalently attached to a 44 kDa HPG scaffold. It was found that peptide density on the conjugate is important for determining biocompatibility and efficacy. Although the antimicrobial activity of aurein 2.2 is improved compared to PEG conjugates, there was a 7-8-fold decrease (16 µg/ml to 110-120 µg/ml) in MIC. Thus further improvements in the activity are desired. In the current study, we therefore investigate strategies to improve the activity of the AMP-HPG bioconjugates, while preserving biocompatibility. We initially used a peptide array, where tryptophan (W) and arginine (R) mutations were introduced, to create more active mutants of aurein 2.2. These more active AMPs were then conjugated to HPGs of different molecular weights (22 kDa to 105 kDa). The antimicrobial activities of the newly identified and most active peptide and its HPG conjugates were measured against S. aureus and compared to the unconjugated peptides. In addition to the biocompatibility measurements, we investigated the resistance against tryptic degradation of the HPG conjugates as compared to the free peptides. Solution CD spectroscopy was used to examine the peptide secondary structure when bioconjugates interacted with model biomembranes in order to understand their mode of action. Overall, these studies aimed to devise improved bioconjugates and to further our understanding of the parameters that are important for obtaining optimal properties. EXPERIMENTAL All chemicals were sourced from Sigma Aldrich Canada Ltd. (Oakville, ON) and used without further purification, except for glycidol, which was purified by vacuum distillation. 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 4


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(POPG) were purchased from Avanti Polar Lipids (Alabaster, AL). Pierce BCA protein assay kit was bought from Thermo Fisher Scientific Inc. (Rockford, IL). Dialysis membrane was obtained from Spectra/Por Biotech (Rancho Dominguez, CA). Peptide array A peptide array with tryptophan (W) and arginine (R) mutations was designed using the aurein 2.2 peptide as the framework. Arrays of peptides were SPOT-synthesized on cellulose membranes by Kinexus Inc. (Vancouver, BC, Canada), as described previously27,28. Each cellulose spot was assumed to have a peptide density of 150 nmol based on Pierce BCA protein assay kit and a purity of ~70%. Peptides were dissolved in 200 µL of autoclaved water and the solution was incubated at room temperature for ~4 h with gentle shaking to solubilize the peptide from the cellulose membrane. The stock peptide solutions were serially diluted and used for the antimicrobial screens described below. From the array, some of the most active peptides (Table 1) were synthesized using solid phase peptide synthesis and purified (95% purity), as described below. The antimicrobial activity of the peptides synthesized in house was verified and found to be consistent with those found for the peptides in the array (Table 1). Peptide synthesis and purification Solid phase peptide synthesis (Fmoc) was used to synthesize aurein 2.2∆3-cys and its RW mutants (Table 1), as previously described29-30. A detailed procedure is reported in the Supporting Information.

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Table 1: Antimicrobial activity (MICs) of selected peptides identified in the peptide array.@ Peptide

MIC S. aureus (µg/mL)

1. GLFDIVKKVVGAL (parent peptide)

32

24. GLWDIWKKWWGWL

16

34. GLFDIVRRVVRAL

16

77. RLWDIVRRVWGWL

4

91. GLWDIWRRWWRWL

8

@

The parent peptide, aurein 2.2, was used as a template. The other peptides in the library were obtained by incorporating arginine (R) and tryptophan (W) residues at various locations. Synthesis of bioconjugates Synthesis of HPG Synthesis and characterization of hyperbranched polyglycerol (HPG) with different molecular weights was carried out as previously reported19,31 (see Supporting Information and Figure S1). The absolute molecular weight and polydispersity of the HPGs were determined using gel permeation chromatography (GPC) coupled with multi angle light scattering (MALS). Amine Modification of HPG HPG was vacuum dried for overnight at 70 °C prior to the reaction. A flame dried round bottom flask was loaded with dried HPG (22 kDa, 200 mg) in anhydrous DMF (20 mL) under Ar. To this solution, NaH (3 eq, w.r.t. targeted -OH groups; 15 mg) was added slowly and stirred at RT for 2 h. To the obtained white cloudy solution, N-glycidylphthalimide (1.5 eq; 54 mg) was added and stirred for another 20 h at 70 °C. The reaction mixture was quenched with dilute HCl (pH ~7) and the resulting reaction mixture was concentrated approximately 3 times the initial concentration. The polymer was purified by precipitation from acetone, followed by dialysis against water (MWCO – 10000 Da, 24 h). 6


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The polymer was lyophilized and the number of phthalimide groups on HPG was determined by 1H NMR (Figure S2). The HPG-phthalimide was deprotected to HPG amine by refluxing it with hydrazine (20 eq, 35 wt. % in H2O; 383mg) in methanol (20 mL) for 24 h. The reduction of HPG-phthalimide was confirmed by 1H NMR (Figure S3) and the resulting HPG amine was purified by dialysis and lyophilized. The number of amine groups was equivalent to phthalimide groups on HPG. Conjugation of peptide with HPG-amine The HPG-amine (0.014g, 22000 g/mol) was reacted with 3-(maleimido)propionic acid Nhydroxysuccinimide ester (0.009g) in dimethylformamide (DMF) at room temperature (22 ºC) for 24 h. The reaction mixture was dialyzed in DMF (MWCO 1000) for 24 h to remove the unreacted 3(maleimido)propionic acid N-hydroxysuccinimide ester. 1H NMR was used to characterize the product (Figure S4). The maleimide modified HPG (0.010g, 22000 g/mol) was reacted with sulfhydryl containing aurein2.2∆3-cys (GLFDIVKKVVGALC-CONH2) (0.007g, 1460.49 g/mol) or its RW mutant (peptide 77c, 0.009g, 1857.34 g/mol) in DMF for 24 hours. The resulting conjugate was purified by size exclusion chromatography (Waters 600 system Mississauga, Ontario, Canada) using a CATSEC column (CATSEC 300, Eprogen)32 and a UV detector (229nm) with a mobile phase composed of 0.5M NaCl in 20% ethanol/water mixture. The product was further characterized by GPC and 1H NMR (Figure S5). The maleimide to thiol-maleimide conversion for 22k 77c and 22k aurein 2.2∆3-cys was 70% and 80%, respectively. The unreacted maleimide was quenched with ethanethiol. The detailed synthesis of the other conjugates can be found in Supporting Information. The conjugates were characterized for their molecular weights by GPC-MALS, conjugation efficiency by 1H NMR and bicinchoninic acid (BCA) protein assay. Details are given in Supporting Information. Synthesis of PEG 77c conjugate The mPEG-maleimide (5000K) was purchased from advanced polymer materials Inc. (Montreal, Canada). The mPEG maleimide (0.01g) was dissolved in 1 mL of acetonitrile and added to sulfhydryl 7



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containing peptide 77c (0.004g, 1857.34 g/mol) dissolved in 1 mL of DMF and stirred for 24 h. The PEG conjugated peptide was purified the same way as the HPG conjugated peptide (described previously) using a size exclusion CATSEC 100 column and characterized by 1H NMR (Figure S6). Antimicrobial activity measurements (minimum inhibitory concentration or MIC ) The MICs of the peptides/conjugates against S. aureus were measured based on previously described methodology19;29,30,33. Details are given in our previous report19 and in Supporting Information. MIC determination after tryptic degradation To determine whether conjugation of the AMP with HPG prevents or alters degradation of the peptides by trypsin, we incubated the peptides/conjugates with trypsin. Trypsin (3 µg) was added to peptide 77c (200 µg/mL solution) and the 22K-77c conjugate (200 µg/mL solution) and incubated at 37 °C for 3 h. After 3 h, the MALDI spectra of the two solutions were acquired. The trypsin-digested samples without purification were used for determination of the MIC of the peptides/conjugates, as described previously. Biocompatibility analysis The biocompatibility of conjugates and peptides were evaluated by measuring the cell viability against

mouse

embryonic

fibroblasts

(3T3-L1)

by

MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-

Diphenyltetrazolium) assay. The blood compatibility of the conjugates were determined by measuring their effect on blood coagulation, effects on platelets, complement activation and red blood cell lysis using human blood. The blood was obtained from unmedicated consenting donors; the protocol was approved by the clinical ethical committee of the University of British Columbia. Detailed protocols are based on our published report19. Detailed procedures are given in Supporting Information.

8


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Biomembrane interaction: CD spectroscopy As previously described29,30,33, a JASCO J-810 spectropolarimeter was used to conduct circular dichroism experiments at 25 ºC. A 1mm cuvette was filled with 200 µL of sample and the spectra were obtained over a wavelength range of 190 nm - 250 nm. A continuous scanning mode was employed with a response time of 1 second, bandwidth of 1.5 nm, 0.5 nm steps, and a scan speed of 200 nm/min. An average of 5 scans was obtained to improve the signal to noise ratio. The spectra were corrected by subtracting the background (e.g. phospholipid spectrum) from the sample spectrum. An average alpha helical content was reported by calculating the output %helix from three fitting programs (CDSSTR, CONTINLL, and SELCON3)34 and averaging these three numbers. RESULTS AND DISCUSSION Discovery of potent peptides using peptide array based on aurein peptide Trytophan and arginine have unique properties that allow them to interact with each other and the bacterial membrane35. The cation-π interactions between the residues allow maximum hydrogen bonding for the arginine side chain and allow the peptide to penetrate deeper into the bacterial membrane35. Using the parent peptide aurein 2.2, with three residues removed from the C-terminus, we designed a peptide array (~100 peptides) with single and multiple W and R mutations. Previous work had shown that truncation of aurein 2.2 by three residues at the C-terminus has minimal impact on its activity33. In order to determine how the mutations impact antimicrobial activity, the sequences were plotted as helical wheels36 (Figure 1). Interestingly, changing single hydrophobic residues, e.g. F or V, for W does not improve activity (Figure 1b). However, adding a W near the interface of the hydrophobic/hydrophilic boundary results in a peptide that is 2x more active. Positioning the tryptophan at this interface may help the peptide anchor into the membrane better, as W is known to snorkel37. Mutation of the lysine residues for arginine (Figure 1c) at single positions (K7 or K8) has a surprising negative impact on activity. The mutation of G11R is most likely beneficial as this introduces a positive 9



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charge in close proximity to the negatively charged D4. Active AMPs typically have multiple positive charges38. From the peptide array, we identified peptide 77 (Figure 1d) that was 8 fold more active (MIC of 4 µg/mL) than the parent peptide (Table 1). A comparison of peptides with multiple mutations (Figures 1d, 1e, 1f) shows that clear trends are difficult to extract. Peptides 77 and 91 have the same number of R residues (and hence the same overall charge), yet peptide 91 is less active (Table 1). The addition of more W residues in peptide 91 does not appear to be beneficial, when comparing to peptide 77. Finally, although peptides 87 and 77 have the same number of mutations, their difference in activity is dramatically different. Overall, an examination of the sequence indicates that both the position and number of R and W residues play a critical role with respect to the activity of AMPs.

a)

b)

c)

d)

e)

f)

x8x4x2x1x2x4x8 less active more active Figure 1: Helical wheels for some of the peptides generated in the array from a) the parent peptide aurein 2.2-∆3. The helical wheel for aurein 2.2-∆3 was generated using the script available at http://rzlab.ucr.edu/scripts/wheel/wheel.cgi. In b), single mutations of W are shown, along with the fold increase/decrease in MIC, as indicated in the legend. In c), single mutations in R are shown. In d)-f), some of the more active peptides are illustrated: d) peptide 77, which is 8x more active than the parent peptide; e) peptide 91, which is 4x more active; and f) peptide 87, which is just as active as aurein 2.2-∆3. 10


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Synthesis of aurein 2.2∆3-cys and peptide 77c conjugates To develop the HPG conjugates, peptides with an additional cysteine at the C terminus were synthesized to facilitate the conjugation (Figure 2). Since the N-terminus is critical for activity of aurein peptides30,33, the C-terminus was chosen for the cysteine addition. The addition of the cysteine residue had minimal to no impact on the antimicrobial activity of the peptides (Table 2).

Figure 2: Synthetic scheme for the conjugation of the peptides with HPG. Only a small number of hydroxyl groups of HPG are shown to ensure clarity. A new amine modification protocol and bifunctional linker was used for accurate quantification of the amine and maleimide groups, respectively. This also ensured water free reactions as peptide 77c was not as readily soluble in water as aurein 2.2∆3-cys.

A general scheme for the HPG-AMP conjugate synthesis is shown in Figure 2. HPGs (22, 44 and 105 k) were synthesized by polymerization of glycidol via anionic ring opening and were modified to generate primary amine functionality39. This was further modified with a bifunctional linker (311



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(maleimido) propionic acid N-hydroxysuccinimide ester) to generate maleimide groups on HPG. Aurein 2.2∆3-cys and peptide 77c were each conjugated to this scaffold via Michael type addition

Table 2: Characteristics of the peptides and the HPG conjugates used in this study@.

Peptide/conjugates

Number of peptide

Molecular

groups per polymer

weight

PDI

MIC

Fold

Number

S.aureus c

decrease

of

in

peptides/

activity

k Da

(95% CId) µg/mL GPC

NMR

aurein 2.2∆3-cys

-

-

1 461a

-

32 (26-38)

1

peptide 77c

-

-

1 858a

-

8 (5-11)

1

22k aurein 2.2∆3-cys

8±2

8±1

33 000b

1.2

85 (74-95)

3

0.24

22k 77c

8±1

7±1

36 000b

1.3

50 (39-61)

6

0.22


16 ± 2

15 ± 2

68 000b

1.4

150 (133-167)

5

0.24

44k 77c

13 ± 1

11 ± 3

67 000b

1.3

100 (83-117)

13

0.19


30 ± 3

26 ± 5

148 000b

1.4

475 (390-560)

15

0.20

105k 77c

25 ± 4

20 ± 5

151 000b

1.4

325 (268-382)

41

0.16

-

1

1.2

125 (102-148)

16

0.20

PEG 5k-77c

@

The number average molecular weights (Mn) HPGs used in this study are 22k (PDI 1.1), 44k (PDI

1.2), and 105k (PDI 1.2). For comparison, a mono-substituted PEG-peptide 77c construct was also synthesized. The number of peptides attached is indicated in brackets in the first column, as determined by GPC analysis. The sequence for aurein 2.2∆3-cys and peptide 77c was GLFDIVKKVVGALC and RLWDIVRRWVGWLC, respectively. a

Molecular weight determined by MALDI-TOF.

b

Molecular weight determined by GPC-MALLS.

c

MICs are the most frequent values observed out of three repeats.

d

95% confidence intervals 12


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of thiol group of the peptide and maleimide groups on HPG. The number of maleimide groups on the polymer was varied for the generation of conjugates with different number of peptides (Table 2). The bioconjugate was purified using size exclusion HPLC and characterized by proton NMR. The NMR data for the precursors and the final conjugates are shown in Figures S1 to S5 in Supporting Information. The molecular weight of the HPG conjugates increased compared to the parent polymer (Table 2) with a molecular weight distribution (PDI) in the range of 1.2 to 1.5. The PDI of the conjugates did not increase considerably compared to the parent HPG suggesting a uniform distribution of the peptides on the polymer molecule. The number of peptides on the conjugates was determined by determining the peptide concentration obtained from a BCA protein kit and the conjugate molecular weight (Table 2). For a given molecular weight scaffold, the number of aurein 2.2∆3-cys and peptide 77c remained similar, and thus comparisons were made for their activity and biocompatibility (see below). Antimicrobial activity of the conjugates Table 2 summarizes the minimal inhibitory concentration (MIC) values for the free peptides and their bioconjugates against clinical Gram positive strain S. aureus (C622). The MIC value of the conjugates is with respect to peptide concentration. The MIC value obtained for 44k aurein 2.2∆3-cys of 150 µg/ml is close to the previously obtained value for the HPG-aurein2.2∆3-cys (5%) of 110 µg/ml19. These two samples contain a similar number of peptides. In terms of fold decrease in activity relative to the free peptide (calculated using the 95% confidence interval values40 in Table 2), this translates into values of 4-5 for the conjugate reported here versus 5-8 for the conjugate in Kumar et al. 19

. This suggests that the difference in conjugation chemistry used here results in a slightly better

conjugate, but the more striking result of this work is that we were able to synthesize a conjugate (22k 77c) with an MIC of 50 µg/mL, which is one of the most active polymer-AMP conjugates reported to date in literature9,10,19. 13



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Looking at the entries in Table 2 in general, the HPGylated peptides showed weaker antimicrobial activity compared to the free peptides. This was expected as polymer conjugation to AMPs is known to decrease their antimicrobial activity9,10,16,19. A few important observations were made based on the MIC values of the conjugates. For both peptides, with an increase in the molecular weight of the polymer scaffold, the antimicrobial activity of the conjugates decreased (Table 2). The activity decrease was slightly more pronounced in the case of peptide 77c conjugates than aurein 2.2∆3cys. This suggests that the peptide sequence plays a crucial role in determining the activity of the conjugates. However, it is important to point out that since peptide 77c is more potent than aurein 2.2∆3-cys its conjugates had overall lower MICs. In practical terms, both the molecular weight and peptide sequence are critical factors that impact the antimicrobial activity of the conjugates. In comparison to PEG 5K 77c, the 22k 77c and 44k 77c are more active (Table 2). The increase in MIC of the conjugates with increasing molecular weights of conjugates could be related to differences in the interaction of the peptides with the polymer chain, to modifications of the secondary structure of the peptide, to its hydrophobicity and solubility, or to changes in the interaction of conjugates with bacterial membrane. Based on the MIC values and peptide density (last column in Table 2), we can state that PEG lowers the activity of the peptide more than HPG upon conjugation. This could be possibly due to the compact structure of HPG compared to PEG32. Our current data as well as literature support this observation. For instance, the decrease in antimicrobial activity of 22k 77c is less prominent when compared to PEGylated AMPs like tachyplesin 1 which showed a 32-64 fold decrease in activity or nisin which became inactive upon PEGylation9,16. It is important to note when comparing the activities of HPGylated and the PEGylated peptide 77c (Table 2) that not only is there a difference in the molecular weight of the polymer, but there is also a difference in the number of peptides attached.

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We also investigated the activity of peptide and conjugates in serum and whole blood. The data is shown in Figure S9A and Figure S10 (supporting information). At the concentrations studied, both the peptide 77c and 22K 77c were effective at clearing the bacteria. Resistance to proteolysis: tryptic degradation Traditionally, many strategies have been employed to limit enzymatic degradation of antimicrobial peptides for their systemic use. These include chemical modification of the peptide (side chain groups, N and C-termini,) incorporation of D amino acids, and cyclization38,41–43. Traditionally polymer conjugation to peptides has increased the stability of peptides against proteolytic degradation 44,45

. In the current work, we determined whether the conjugation of peptide 77c to HPG prevents the

peptides from protease degradation. We incubated peptide 77c and 22K 77c with trypsin for 3 h and characterized the peptide fragments generated by MALDI-TOF mass spectrometry. We have seen that the conjugated peptide (22K 77c) did not show characteristic cleavage of the naked peptide 77c (characteristic m/z 1114, 988) when incubated with trypsin (Figure 3), suggesting that the conjugated peptide 77c on HPG is resistant to tryptic degradation. The additional peaks present in the MALDI spectra were from the matrix, as can be seen from the MS spectrum in Figure S11. In order validate that the degradation of the peptides leads to loss in antimicrobial activity, the MIC of the samples treated with trypsin were determined. Peptide 77c was inactive once treated with trypsin for 3 hours, whereas 22K 77c retained the same MIC (50 µg/mL) before and after trypsin treatment. This further indicates that the peptide is protected from protease degradation once conjugated to HPG and maintains activity. Trypsin alone did possess any bactericidal activity. Peptide secondary structure on bioconjugates To further investigate the mechanism of action of HPG-conjugates, we determined the secondary structure of the conjugates using CD spectroscopy. Typically, AMPs do not adopt secondary 15



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structure in an aqueous environment but fold upon interaction with a lipid membrane. Initially, positively charged residues interact with the negatively charged head groups found in the outer leaflet of the membrane bilayer, followed by thermodynamically favorable hydrophobic-hydrophobic interactions between the AMP and the interfacial membrane lipids, leading to the formation of a specific conformation46–48. In our study, both free peptides are random coil in phosphate buffer (Figure 4a). Interestingly, peptide 77 adopts an alpha helical conformation once conjugated to HPG in phosphate buffer without the presence of phospholipids or mimics such as trifluoroethanol (TFE). All molecular weight conjugates (22k 77c, 44k 77c and 105k 77c) of 77c adopt alpha helical structure in phosphate buffer, which indicates it is an intrinsic property of the peptide-polymer conjugate. In the presence of phospholipids, the percent helicity of the 77c conjugates increases indicating a more favorable conformation upon interaction with a membrane mimetic (Table 3, Figure 4). However, the increase in helicity is much smaller for the conjugates than the free peptide in the presence of POPC/PG (1:1) relative to PBS. On the other hand, once HPGylated with various molecular weight HPG, aurein 2.2∆3-cys remains as a random coil. It only adopts an alpha helical structure in the presence of phospholipids. Compared to the free peptide, the percent helicity for the HPGylated aurein 2.2∆3-cys is much lower

16


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Figure 3: MALDI-TOF mass spectra of a) 77c, b) 22k 77c and c) trypsin after 3 hours of digestion. Trypsin was added to the peptides/conjugates and incubated at 37 °C for 3 hours, followed by MALDI characterization. Trypsin cleaves after positively charged residues such as arginine as shown in a). The peptide fragments detected for peptide 77c are highlighted in the red box in a). 17



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Figure 4: CD Spectra of the conjugates in a) phosphate buffer and b) POPC/PG (1:35). The peptides and conjugates have a higher helical content in the presence of lipids. Interestingly, conjugation of peptide 77c to HPG results in more structured peptides. 18


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Table 3: Alpha helical content (%) of the various conjugates studied here, as determined from fits from the CD spectra (Figure 4). PBS

POPC/POPG (1:1) Peptide/Lipid (1:35)

aurein 2.2∆3-cys

7

97


8

47


4

40


5

30

peptide 77c

8

73

22k 77c

40

65

44k 77c

31

52

105k 77c

23

41

than the free aurein 2.2∆3-cys, which is similar to previous studies19. HPGylation of aurein 2.2∆3-cys peptide does not affect the structure of the peptide, whereas HPGylation of the peptide 77c enhances the secondary structure of the peptide in buffer. However, HPGylation of both peptides leads to a decrease in percent helicity in a membrane-like environment when compared to the respective free peptides (Table 3). Finally, the data in Table 3 clearly shows that the helicity for a given peptide conjugate decreases with increasing HPG molecular weight. In this case, the alpha helical content correlates well with the activity (Table 2). Cell compatibility of the peptides and conjugates The biocompatibility of the antimicrobial peptides and their conjugates is crucial for their use in various applications49–51. Therefore, the cell viability was assessed against human fibroblasts by the MTT assay. It is known from previous studies that aurein 2.2∆3-cys is cytotoxic to fibroblasts19. It 19



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causes a decrease in cell viability at a concentration of 62.5µg/mL (Figure 5a). However, once conjugated to HPG (22k, 44K or 105k), its cell tolerance is increased irrespective of the molecular weight of the HPG used (Figure 5a). Peptide 77c was also toxic towards fibroblast at 2x the MIC concentrations (15.62 µg/mL). However, once HPGylated, peptide 77c (22k 77c) showed better tolerance even at concentrations of 5x the MIC (250 µg/mL). All the HPGylated peptides showed the same trend irrespective of the molecular weight of the scaffold. It is possible that the large HPG moiety prevents the HPGylated peptides from permeating through the extracellular matrix effectively, because of steric hindrance. This phenomenon has been previously observed for PEGylated magainin, tachyplesin, and a synthetic AMP CaLL9,10,52. Our biomembrane interaction data (Table 3 and Figure 4) support this statement; there was a significant decrease in the secondary structure formation upon conjugation of the peptides to HPG. Blood compatibility of the peptide and peptide conjugates Although the conjugates are compatible with cultured cells, it is important to determine the interaction with blood, as this conjugates will be potentially used in systemic administration. There are only a few reports in the literature on the interaction of cationic AMPs and/or conjugates14,53 with various blood components. Moreover, researchers are actively seeking to develop peptides that minimize red blood cell lysis, as described in a recent study that used computational methods54. Given this importance, we sought to understand whether the attachment of two different (in terms of activity) cationic AMPs on HPG (with different molecular weight) would alter their blood interaction.

20


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Percent Viability

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Me

o dia

nly

Au

n rei

2.2 k 22

ys 3-c n rei u A

2.2

ys 3-c

kA 44

u

n rei

2.2 5k 10

ys 3-c A

in u re

2.2

ys 3-c

Figure 5: Fibroblast cell viability upon exposure to free peptides and HPG peptide conjugates. a) aurein 2.2∆3-cys *P=0.0056; **P=0.2121. b) peptide 77c *P=0.0047; **P=0.2007. Viability of the cell after incubation with free peptides and peptides conjugated to HPG at various concentrations for 48 h at 37 °C. 21



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Platelet activation, red cell lysis, complement activation and blood coagulation were used to test the interaction of HPG conjugates with different blood components. For all the experiments volume ratio of blood/plasma to peptide/conjugate was kept at 9:1 v/v. Washed RBCs were incubated with the peptide/conjugates and the percent lysis was measured in order to investigate the interaction of conjugates with RBCs. Aurein 2.2∆3-cys causes about 10% RBC lysis at 2x the MIC concentration (62.5 µg/mL), whereas 22k HPG-aurein 2.2∆3-cys (MIC 85 µg/mL) was not cytotoxic at nearly 3x its new MIC concentration (250 µg/mL, Figure 6). Peptide 77c caused similar RBC lysis compared to aurein 2.2∆3-cys at 2x the MIC. At 16 µg/mL (2x the MIC), peptide 77c also caused about 10% RBC lysis, however once conjugated to 22k polymer (22K 77c, MIC 50 µg/mL), the conjugate cause 10% lysis at 5x the new MIC (250 µg/mL). The data suggests that neither the activity of the peptide nor the polymer molecular weight influence the RBC lysis. The hydrophobic masking effect and charge of HPG work universally, contributing to the diminished toxicity of all the conjugates towards RBCs. A very similar observation was made upon polymer conjugation of other AMPs, such as KYE2814 and CaLL52. Platelets are important players in the blood clotting cascade. Many thrombotic complications, such as the formation of blood clots inside blood vessels, can arise when platelets are activated. Cationic peptides/polymers have been shown to activate platelets22. Thus we examined whether the peptides/conjugates induce platelet activation in human PRP. Expressed glycoprotein P-selectin CD62 on the surface of platelets by fluorescently labeled monoclonal anti-CD62P-FITC antibody and flow cytometry was used to measure platelet activation. Aurein 2.2∆3-cys and all of its various molecular weight conjugates do not activate platelets at the concentrations indicated (Figure 7). Interestingly, peptide 77c on its own activates platelets at 250 µg/mL (30% activation), but none of the HPGylated 77c conjugates activate platelets. Again, this suggests that HPG has the ability to mask the negative effects of the peptides, independent of HPG molecular weight. 22


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Figure 6: Red blood cell lysis upon exposure to free peptides and HPG peptide conjugates: a) aurein 2.2∆3-cys *P=0.0075; **P=0.5469. b) peptide 77c *P=0.0004; **P=0.0122. Hemolysis of free peptides and HPG conjugates was measured at various concentrations, after 1h incubation at 37 °C with washed red blood cells. PBS buffer was used as normal control. 23



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(a) 15.62 µg/mL

50

31.25 µg/mL 40

62.5 µg/mL 125 µg/mL

30

250 µg/mL

20 10 0 S PB A

ei n ur

2.2 k 22

ys 3-c A

in ure

2.2

ys 3-c

kA 44

2 in u re

.2

5k 10

ys 3-c n rei Au

2.2

ys 3-c

Figure 7: Platelet activation upon exposure to free peptides and HPG peptide conjugates: a) aurein 2.2∆3-cys; b) peptide 77c. *P=0.0094; **P=0.5944. Human platelet rich plasma was used to measure platelet activation. Platelets that expressed the activation marker CD62P using the monoclonal anti-CD62-PE antibody were counted (flow cytometry). PBS buffer was used as a negative control.

24


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The complement system is an important part of innate immunity. As many as 30 different proteins are part of this system. Complement activation upon interaction with the peptides and/or the conjugates is an indication of inflammatory potential such as histamine release and suggests blood incompatibility. Complement activation was determined by a complement consumption analysis using a antibody-sensitized sheep erythrocyte complement lysis (CH50) assay using human serum55 to determine whether the free and HPGylated peptides had an effect on the complement system. Neither aurein 2.2∆3-cys, peptide 77, nor the HPGylated peptides activate complement (Figure 8), indicating that they are all blood compatible. Clinical coagulation assays were used to study the blood coagulation profile of the peptide and conjugates56. This test provides important information on the pro- or anti-coagulant activities of the peptides/conjugates. Blood coagulation was measured by clinical coagulation assays activated partial thromboplastin time (aPTT) and partial thromboplastin time (PT) in human plasma. In aPTT measurements, clotting time is taken as the time it takes for fibrin clot to form once thromboplastin reagent and calcium chloride is added. Aurein peptides were shown to influence the blood coagulation by delaying the onset of coagulation19. Both peptides and the conjugates did not significantly affect the aPTT values when compared to the HEPES-buffered saline (HBS) control (Figure S7) (supplementary information). Prothrombin time (PT) was measured to characterize the extrinsic pathway of blood coagulation upon addition of free peptides and conjugates. Cationic peptides and polymers have been shown to delay prothrombin time22,31. Interestingly, peptide 77c has no impact on PT whereas aurein 2.2∆3-cys prolonged PT at concentrations as low as 125 µg/mL, suggesting that peptide 77c is not changing the blood coagulation profile compared to aurein 2.2∆3-cys. None of the conjugates showed any changes in the PT (Figure 9). In the conjugates, HPG may mask the hydrophobic nature of the peptides and prevent

25



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Figure 8: Complement activation upon exposure to free peptide and HPG peptide conjugates: a) aurein 2.2∆3-cys; b) peptide 77c. A complement consumption assay (CH50) was used to determine complement activation, using antibody-sensitized sheep red blood cells and human serum. The percentage of consumed complement proteins is shown. PBS buffer was used as a normal control.

26


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Figure 9: Prothrombin time upon exposure to free aurein peptide and HPG peptide conjugates: a) aurein 2.2∆3-cys *P=0.0067; **P=0.2803. b) peptide 77c. Prothrombin time (PT) was measured by incubating peptide and conjugates with platelet poor plasma at 37 °C. 27



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non-specific interactions with blood coagulation proteins, which leads to normal prothrombin time. To further investigate the influence of our peptide and its conjugates on whole blood clotting profile, we used rotational thromboelastometry (ROTEM) measurements in human whole blood. Results are shown in Figure S8a and S8b. Representative ROTEM traces are shown in Figure S8a and clotting time is shown in Figure S8b. Both the peptide and conjugate (peptide 77c and 22K 77c) showed similar clotting profile as that buffer control suggesting that the conjugate or peptide is not altering the blood coagulation profile. The clotting time of the peptide and conjugates is also not significantly different from the buffer control under these conditions. CONCLUSIONS In this manuscript, we examined how the parameters such as peptide sequence and size of HPG impact the antimicrobial activity and blood compatibility of AMP/HPG bioconjugates. Specifically, we designed a R and W mutant peptide library derived from the previously studied aurein 2.2 peptide and obtained peptide 77 that was 4-8 fold more active compared to the parent peptide. We conjugated both aurein 2.2 and the new peptide to HPGs of varied molecular weight and examined the effect of HPGylation on the antimicrobial activity, structure and interaction with blood components. Overall, HPGylation of both peptides decreased the antimicrobial activity, with the best molecular weight being 22k, where a factor of 3-6 in the decrease in MICs was observed. More importantly, we obtained a bioconjugate with the best MIC value reported in literature to date, namely 50 µg/ml. This bioconjuate (22K 77c) also showed resistance to tryptic digestion and maintained antimicrobial activity compared to the free peptide. HPGylation decreased the alpha helical content of both peptides in a membrane environment; however, in buffer HPGylated peptide 77c adopted an alpha helical structure compared to HPGylated aurein 2.2∆3-cys which was random coil. Even though the activity and structure of the peptides are different, HPGylation reduced the interaction with red blood cells and platelets. All HPGylated peptides were biocompatible with fibroblast cells, demonstrating their non-toxic behavior 28


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compared to the free peptides. The results presented here demonstrate that it is possible to optimize peptide sequence and HPG molecular weight to obtain bioconjugates with excellent properties. The results suggest that conjugating highly active peptides with low molecular weight HPG is the best strategy.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS The authors would like to acknowledge funding from the following agencies: Natural Sciences and Engineering Research Council of Canada (NSERC) and Canadian Institutes of Health Research (CIHR), in the form of CHRP funding; Canada Foundation of Innovation (CFI) and the British Columbia Knowledge Development Fund (BCKDF) for funding the NMR spectrometer used in this study. The infrastructure facility at Centre for Blood Research (CBR) is supported by the CFI and BCKDF. JNK is the recipient of a Michael Smith Foundation for Health Research (MSFHR) Career Investigator Scholar award. We also thank the following individuals: Fred Rossell of the CD facility in the CBR at UBC for maintaining the instrument; Evan Haney from the Hancock lab for initial guidance with the peptide array; Irina Chafeeva for help in GPC studies; and the Overall lab at CBR for maintaining the MALDI instrument. Finally, SA acknowledges a MSFHR postdoctoral fellowship. UA and MTK each acknowledge a graduate scholarship from Centre for Blood Research.

SUPPORTING INFORMATION. Detailed description of the experimental protocols are given including peptide and HPG synthesis, amine modification, and conjugation for the various HPG 29



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molecular weights, antimicrobial activity determination and biocompatibility measurements. Description of combined blood lysis and bacteria killing assay and bacteria killing assay in serum. Description of rotational thromboelastometry. Synthetic scheme for HPG (Figure S1). 1H NMR spectra used to characterize 22k HPG at various steps in the synthesis (Figures S2-S5). 1H NMR spectrum of the 5k PEG 77c bioconjugate (Figure S6). Influence of peptide and peptide conjugates on activated Partial Thromboplastin Time (aPTT) (Figure S7). Thromboelastometry data (Figures S8a and b). RBC lysis data for blood in the presence of bacteria (Figures 9a and 9b). Bacterial killing in the presence of serum (Figure S10). MALDI mass spectrum of the matrix (Figure S11).

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Antimicrobial PeptideâPolymer Conjugates with High Activity

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